Insertion of a neomycin selection cassette in the Amigo1 locus alters gene expression in the olfactory epithelium leading to region-specific defects in olfactory receptor neuron development.

During development of the nervous system, neurons connect to one another in a precisely organized manner. Sensory systems provide a good example of this organization, whereby the composition of the outside world is represented in the brain by neuronal maps. Establishing correct patterns of neural circuitry is crucial, as inaccurate map formation can lead to severe disruptions in sensory processing. In rodents, olfactory stimuli modulate a wide variety of behaviors essential for survival. The formation of the olfactory glomerular map is dependent on molecular cues that guide olfactory receptor neuron axons to broad regions of the olfactory bulb and on cell adhesion molecules that promote axonal sorting into specific synaptic units in this structure. Here, we demonstrate that the cell adhesion molecule Amigo1 is expressed in a subpopulation of olfactory receptor neurons, and we investigate its role in the precise targeting of olfactory receptor neuron axons to the olfactory bulb using a genetic loss-of-function approach in mice. While ablation of Amigo1 did not lead to alterations in olfactory sensory neuron axonal targeting, our experiments revealed that the presence of a neomycin resistance selection cassette in the Amigo1 locus can lead to off-target effects that are not due to loss of Amigo1 expression, including unexpected altered gene expression in olfactory receptor neurons and reduced glomerular size in the ventral region of the olfactory bulb. Our results demonstrate that insertion of a neomycin selection cassette into the mouse genome can have specific deleterious effects on the development of the olfactory system and highlight the importance of removing antibiotic resistance cassettes from genetic loss-of-function mouse models when studying olfactory system development.

Comparative analyses of the Smith-Magenis syndrome protein RAI1 in mice and common marmoset monkeys.

Retinoic acid-induced 1 (RAI1) encodes a transcriptional regulator critical for brain development and function. RAI1 haploinsufficiency in humans causes a syndromic autism spectrum disorder known as Smith-Magenis syndrome (SMS). The neuroanatomical distribution of RAI1 has not been quantitatively analyzed during the development of the prefrontal cortex, a brain region critical for cognitive function and social behaviors and commonly implicated in autism spectrum disorders, including SMS. Here, we performed comparative analyses to uncover the evolutionarily convergent and divergent expression profiles of RAI1 in major cell types during prefrontal cortex maturation in common marmoset monkeys (Callithrix jacchus) and mice (Mus musculus). We found that while RAI1 in both species is enriched in neurons, the percentage of excitatory neurons that express RAI1 is higher in newborn mice than in newborn marmosets. By contrast, RAI1 shows similar neural distribution in adult marmosets and adult mice. In marmosets, RAI1 is expressed in several primate-specific cell types, including intralaminar astrocytes and MEIS2-expressing prefrontal GABAergic neurons. At the molecular level, we discovered that RAI1 forms a protein complex with transcription factor 20 (TCF20), PHD finger protein 14 (PHF14), and high mobility group 20A (HMG20A) in the marmoset brain. In vitro assays in human cells revealed that TCF20 regulates RAI1 protein abundance. This work demonstrates that RAI1 expression and protein interactions are largely conserved but with some unique expression in primate-specific cells. The results also suggest that altered RAI1 abundance could contribute to disease features in disorders caused by TCF20 dosage imbalance.

Spatiotemporal expression of IgLON family members in the developing mouse nervous system.

Differential expression of cell adhesion molecules in neuronal populations is one of the many mechanisms promoting the formation of functional neural circuits in the developing nervous system. The IgLON family consists of five cell surface immunoglobulin proteins that have been associated with various developmental disorders, such as autism spectrum disorder, schizophrenia, and major depressive disorder. However, there is still limited and fragmented information about their patterns of expression in certain regions of the developing nervous system and how their expression contributes to their function. Utilizing an in situ hybridization approach, we have analyzed the spatiotemporal expression of all IgLON family members in the developing mouse brain, spinal cord, eye, olfactory epithelium, and vomeronasal organ. At one prenatal (E16) and two postnatal (P0 and P15) ages, we show that each IgLON displays distinct expression patterns in the olfactory system, cerebral cortex, midbrain, cerebellum, spinal cord, and eye, indicating that they likely contribute to the wiring of specific neuronal circuitry. These analyses will inform future functional studies aimed at identifying additional roles for these proteins in nervous system development.

Kirrel2 is differentially required in populations of olfactory sensory neurons for the targeting of axons in the olfactory bulb.

The formation of olfactory maps in the olfactory bulb (OB) is crucial for the control of innate and learned mouse behaviors. Olfactory sensory neurons (OSNs) expressing a specific odorant receptor project axons into spatially conserved glomeruli within the OB and synapse onto mitral cell dendrites. Combinatorial expression of members of the Kirrel family of cell adhesion molecules has been proposed to regulate OSN axonal coalescence; however, loss-of-function experiments have yet to establish their requirement in this process. We examined projections of several OSN populations in mice that lacked either Kirrel2 alone, or both Kirrel2 and Kirrel3. Our results show that Kirrel2 and Kirrel3 are dispensable for the coalescence of MOR1-3-expressing OSN axons to the most dorsal region (DI) of the OB. In contrast, loss of Kirrel2 caused MOR174-9- and M72-expressing OSN axons, projecting to the DII region, to target ectopic glomeruli. Our loss-of-function approach demonstrates that Kirrel2 is required for axonal coalescence in subsets of OSNs that project axons to the DII region and reveals that Kirrel2/3-independent mechanisms also control OSN axonal coalescence in certain regions of the OB.

Loss of Kirrel family members alters glomerular structure and synapse numbers in the accessory olfactory bulb.

The accessory olfactory system controls social and sexual behaviours in mice, both of which are critical for their survival. Vomeronasal sensory neuron (VSN) axons form synapses with mitral cell dendrites in glomeruli of the accessory olfactory bulb (AOB). Axons of VSNs expressing the same vomeronasal receptor (VR) converge into multiple glomeruli within spatially conserved regions of the AOB. Here, we have examined the role of the cell adhesion molecule Kirrel2 in the formation of glomeruli within the AOB. We find that Kirrel2 expression is dispensable for early axonal guidance events, such as fasciculation of the vomeronasal tract and segregation of apical and basal VSN axons into the anterior and posterior regions of the AOB, but is necessary for glomeruli formation. Specific ablation of Kirrel2 expression in VSN axons results in the disorganization of the glomerular layer of the posterior AOB and in the formation of fewer and larger glomeruli. Furthermore, simultaneous ablation of Kirrel2 and Kirrel3 expression leads to a loss of morphologically identifiable glomeruli in the AOB, reduced excitatory synapse numbers, and larger presynaptic terminals. Taken together, our results demonstrate that Kirrel2 and Kirrel3 are essential for the formation of glomeruli and suggest they contribute to synaptogenesis in the AOB.

Slitrk1 is localized to excitatory synapses and promotes their development.

Following the migration of the axonal growth cone to its target area, the initial axo-dendritic contact needs to be transformed into a functional synapse. This multi-step process relies on overlapping but distinct combinations of molecules that confer synaptic identity. Slitrk molecules are transmembrane proteins that are highly expressed in the central nervous system. We found that two members of the Slitrk family, Slitrk1 and Slitrk2, can regulate synapse formation between hippocampal neurons. Slitrk1 is enriched in postsynaptic fractions and is localized to excitatory synapses. Overexpression of Slitrk1 and Slitrk2 in hippocampal neurons increased the number of synaptic contacts on these neurons. Furthermore, decreased expression of Slitrk1 in hippocampal neurons led to a reduction in the number of excitatory, but not inhibitory, synapses formed in hippocampal neuron cultures. In addition, we demonstrate that different leucine rich repeat domains of the extracellular region of Slitrk1 are necessary to mediate interactions with Slitrk binding partners of the LAR receptor protein tyrosine phosphatase family, and to promote dimerization of Slitrk1. Altogether, our results demonstrate that Slitrk family proteins regulate synapse formation.

Cellular and molecular mechanisms regulating embryonic neurogenesis in the rodent olfactory epithelium.

Mechanisms that regulate cellular differentiation in developing embryos are maintained across multiple physiological systems, including the nervous system where neurons and glia are generated. The olfactory epithelium, which arises from the olfactory pit, is a stratified tissue in which the stepwise generation of neurons and support cells can easily be assessed and followed during embryogenesis and throughout adulthood. During olfactory epithelium morphogenesis, progenitor cells respond to factors that control their proliferation, survival, and differentiation in order to generate olfactory receptor neurons that detect odorants in the environment and glia-like sustentacular cells. The tight temporal regulation of expression of proneural genes in dividing progenitor cells, including Mash-1, Neurogenin-1, and NeuroD1, plays a central role in the production of olfactory receptor neurons. Multiple factors that either positively or negatively affect the generation of olfactory receptor neurons have been identified and shown to impinge on the transcriptional regulatory network in dividing progenitor cells. Several growth factors, such as FGF-8, act to promote neurogenesis by ensuring survival of progenitor cells that will give rise to olfactory receptor neurons. In contrast, other molecules, including members of the large TGF-ß family of proteins, have negative impacts on neurogenesis by restricting progenitor cell proliferation and stalling their differentiation. Since recent reviews have focused on neurogenesis in the regenerating adult olfactory epithelium, this review describes neurogenesis at embryonic stages of olfactory epithelium development and summarizes our current understanding of how both cell intrinsic and extrinsic factors control this process.